Exhaustion of CD8 cells is a critical problem that limits both vaccine induced, and the natural immune response to cancer. Treatments that rescue exhausted CD8 cells such as anti-PD1 generate a powerful anti-tumor immune response, suggesting it is important to understand the mechanisms controlling this process. In this proposal we show that CD8 T-cells isolated from tumors of mouse models and human prostate cancer patients have an exhausted phenotype. Furthermore, we identified DNA methylation changes in the promoter regions of key molecules associated with exhaustion. Aim 1 (K99) will expand upon our initial epigenetic characterization by performing whole genome analysis of DNA methylation in human and mouse CD8 T-cell subsets. In addition to the epigenetic changes that occur during exhaustion, preliminary data presented here shows that without the DNA methylation enzyme, DNMT3a, T-cells do not exhaust. This suggests that DNA methylation is critical to the process of T-cell exhaustion. In Aim 2 (K99) we will investigate if DNMT3a controls CD8 T-cell exhaustion in prostate cancer models. To do this we will use the DNMT3a knockout mice TrampC2. The production of cytokines, proliferation, and control of tumor growth will all be assessed in this aim. Also, we will define the specific genome wide changes controlled by DNMT3a using whole genome bisulfite analysis (R00). Finally, our data shows that DNMT3a causes DNA methylation changes at precise locations throughout the genome. We show here that the locations where these DNMT3a specific changes occur share high sequence homology to non-coding RNA. Furthermore, we show that DNMT3a binds these non-coding RNA. Based on this data, Aim 3 of this proposal will investigate if lncRNA controls DNMT3a enzyme activity at locations undergoing methylation changes in exhausted CD8 T-cells. This aim will use a lentiviral system to determine if manipulating expression of non-coding RNA can be used to cause specific changes in DNA methylation. We will then perform in vitro and in vivo assays on CD8 T-cells to see if this approach can be used to alter CD8 T-cell function (R00). Finally, we will integrate data collected about the specific methylation events controlled by DNMT3a with lncRNA data collected to discern which non-coding RNAs control DNMT3a dependent epigenetic program in exhausted CD8 T-cells (R00). Together, these aims will determine the epigenetic features of exhausted T-cells derived from prostate cancer, the specific epigenetic program controlled by DNMT3a during exhaustion, and if lncRNA can be used to modulate the epigenetic program acquired by exhausted CD8 T-cells in cancer.